The invention relates to new genes isolated from Neisseria meningitidis. Isolated nucleic acids, probes, expression cassettes, polypeptides, antibodies, immunogenic compositions, antisense nucleic acids, amplification mixtures and new invasion deficient strains of Neisseria meningitidis. The invention also relates to methods of detecting Neisseria meningitidis and Neisseria meningitidis nucleic acids, and to methods of inhibiting the invasion of mammalian cells by Neisseria meningitidis. 
Neisseria meningitidis, a Gram-negative encapsulated diplococcus, is an obligate human pathogen and the causative agent of meningococcal meningitis, one of the most devastating forms of meningitis. These bacteria are isolated from humans worldwide and can cause sporadic and epidemic disease. Person-to-person transfer of N. meningitidis occurs mainly via the airborne route, and is particularly a problem in places where people are in close quarters, such as prisons, military camps, school class rooms, and day care centers. At any one time, between 2 and 10% of individuals in the population carry this organism asymptomatically (Greenfield, S., et al. (1971), J. Infec. Dis., 123:67-73; Moore, P. S., et al. (November 1994), Scientific American, p38-45; Romero, J. D. et al. (1994), Clinical Microbiology Review, 7:559-575). With such a high carrier rate, the threat or potential for outbreaks or epidemics is always present. Although significant advances have been made in the area of the pathogenesis of the organism, there is much to be learned about the genetics and cell biology of the host-parasite interaction.
Understanding the mechanism(s) of attachment and invasion is one of the most important aspects in N. meningitidis disease. In order to cause disease, meningacocci must survive and colonize the mucosa of the nasopharynx, pass through these tissue into the bloodstream replicate to large numbers in the blood, cross the blood-brain barrier and multiply in the cerebrospinal fluid (CFS) where they cause inflammation of the meninges. Various models have been used in order to mimic the events that take place during infection in humans. Mouse models (Miller, C. P. (1933), Science, 78:340-341; Holbein, B. E. (1981), Can. J. Microbiol., 27:738-741; Salit, I. E. (1984), Can. J. Microbiol., 30:1022-1029), human nasopharyngeal organ culture (Stephens, D. S., et al. (1991), Rev Infect Dis., 13:22-33), chick embryo (Buddingh, G. J. et al. (1987), Science, 86:20.21; Pine, L., et al., Micrbiol. Lett., 130:37-44), and tissue culture monolayer and bilayer systems (Birkness, K. A., et al. (1995), Infect. Immun., 63:402-409) represent some of the models commonly used to study virulence of N. meningitidis. 
The organ culture system has been used successfully to assess the attachment and invasion properties of various N. meningitidis strains (Salit, I. E. (1984), Can. J. Microbiol., 30:1022-1029).
Designated by serogroup, serological classification of N. meningitidis is based on the capsular polysaccharide composition of the particular strain. Among the meningococci there are at least thirteen different serogroups: A, B, C, 29-E, H, I, X, L, W135, X, Y and Z. Of these serogroups, A, B and C comprise over 90% of the strains isolated from patients afflicted with meningococcal meningitis (Poolman, J. T., et al. (1995), Infectious Agents and Disease, 4:13-28). The nature of the capsule in serogroups A and C has led to the development of useful vaccines against these serogroups. However, the serogroup B capsular polysaccharide does not induce protection in humans. Many laboratories around the world are concentrating their efforts on the study and characterization of epitopes from various membrane and other extracellular factors for use as vaccine candidates. Some of the most common non-capsule factors in such studies include a number of outer membrane proteins (OMP) such as class 1 (Por A, a cation Specific porin), class 2 or 3 (Pot B, an anion specific protein) and to a lesser extent class 4 and class 5 OMPs (Rmp, and Opc and Opa opacity associated proteins, respectively). While class 5 Opc and Opa OMPs have been shown to play roles in the invasion of epithelial cells (Virji, M., et al. (1992), Mol. Microbiol., 6;2786-96) due to their antigenic and phase variability (Aho, E. L.; et al. (1991), Mol. Microbiol., 5:1429-37), they are not considered to be good vaccine candidates.
Class 1 OMPs appear to be good candidates for vaccine studies since these proteins have been shown to induce protective immunity. Evaluation of various non-capsular antigens as potential vaccine candidates in in vitro bactericidal assays and an infant rat model revealed that class 1 OMP had the highest protective capacity compared to factors such as LPS and class 2/3 OMPs (Saukkonen, K., et al. (1989), Vaccine, 7:325-328). However, preliminary data from vaccine trial studies suggests that these factors do not elicit a complete immune response, especially in children (Romero. J. D. et al. (1994), Clinical Microbiology Review, 7:559-575; Poolman, J. T., et al. (1995), Infectious Agents and Disease, 4:13-28). The development of fusion or hybrid genes containing epitopes from class 1 OMP show great promise as vaccine candidates (Van der Ley, P., et al. (1992), Infect. Immun., 60:3156-3161; Van der Ley, P., et al. (1993), Infect. Immun., 61:4217-4224). However, these hybrids do not elicit protection in infants, and the immunity induced is type specific and very short-lived (Poolman, J. T., et al: (1995), Infectious Agents and Disease, 4:13-28). Far these and other reasons, it is or importance to identify alternative serogroup B vaccine antigens. Initial attachment and invasion by the pathogen is critical to the disease process. If mucosal immunity can be derived against these bacterial factors, the disease process and the carrier state can be prevented. The present invention provides these and other features.
The invention provides nucleic acids and encoded polypeptides associated with invasion of Neisseria meningitidis. The polypeptides are used as diagnostic reagents as immunogenic reagents; and as components of vaccines. The nucleic acids are used as diagnostic reagents, as components of vectors and vaccines, and to encode the polypeptides of the invention. The invention also provides strains of Neisseria meningitidis which have an invasion deficient phenotype.
In one embodiment, the invention provides isolated nucleic acids encoding the polypeptides of the invention, including ORF 1 (SEQ ID NO:2), ORF 2 (ORF2 a (SEQ ID NO:4) and ORF2b (SEQ ID NO:5), two separate embodiments depending on alternate start sites for the ORF2 polypeptide), ORF 3 (SEQ ID NO:7) and, conservatively modified variations of each of the polypeptides. Exemplar nucleic acids include Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), and Seq 3 (SEQ ID NO:7) (see, FIGS. 5, 6, and 7 respectively). Other nucleic acids encoding the same polypeptides include those with silent codon substitutions relative to Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3) for Seq 3 (SEQ ID NO:6); as well as conservatively modified variations thereof.
Isolated nucleic acids which hybridize under stringent conditions to the exemplar nucleic acids Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), or Seq 3 (SEQ ID NO:6) are also provided. For example, a complementary nucleic acid to a sequence provided by Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), or Seq 3 (SEQ ID NO:6) hybridizes to Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), or Seq 3 (SEQ ID NO:6), respectively. Nucleic acids which include substantial subsequences complementary to Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), or Seq 3 (SEQ ID NO:6) also hybridize to Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), or Seq 3 (SEQ ID NO:6), respectively.
Isolated nucleic acids which hybridize under stringent conditions to Seq 4 (SEQ ID NO:8) are provided. Seq 4 (SEQ ID NO:8) is a genomic sequence which encodes Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), and Seq 3 (SEQ ID NO:6). Thus, complementary nucleic acids to sequences provided by Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), Seq 3 (SEQ ID NO:6), or Seq 4 (SEQ ID NO:8) all hybridize to Seq 4 (SEQ ID NO:8) under stringent conditions. Similarly, nucleic acids which include substantial subsequences of Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), Seq 3 (SEQ ID NO:6) or Seq 4 (SEQ ID NO:8) also hybridize to Seq 4 (SEQ ID NO:8). The isolated nucleic acids are optionally vector nucleic acids which comprise a transcription cassette. The transcription cassette optionally encodes a polypeptide. Typically, the portion of the transcription cassette which encodes the polypeptide hybridizes to Seq 4 (SEQ ID NO:8) under stringent conditions. Upon transduction of the transcription cassette into a cell, an mRNA which hybridizes to Seq 4 (SEQ ID NO:8) under stringent conditions is produced. The mRNA is translated in the cell into a polypeptide such as the ORF 1 (SEQ ID NO:2), ORF 2a (SEQ ID NO:4), ORF 2b (SEQ ID NO:5) or ORF 3 (SEQ ID NO:7) polypeptides.
Polypeptides encoded by nucleic acids which hybridize under stringent conditions to Seq 4 (SEQ ID NO:8), including Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), Seq 3 (SEQ ID NO:7) are provided herein. Exemplar polypeptides include ORF 1 (SEQ ID NO:1), ORF 2a (SEQ ID NO:4), ORF 2b (SEQ ID NO:5), or ORF 3 (SEQ ID NO:6).
Full length polypeptides of the invention, or antigenic epitopes derived from the full length polypeptides of the invention are optionally present in immunogenic compositions. The antigenic epitopes are optionally incorporated into fusion proteins which optionally include antigenic epitopes from related or unrelated proteins. The antigenic epitopes are optionally expressed on the surface or antigenic viral vectors.
The immunogenic compositions optionally comprise components to enhance immunogenicity, Such as an adjuvant. The compositions optionally include pharmaceutically acceptable excipients. When administered to a mammal, the immunogenic compositions optionally provide an immune response against antigenic epitopes which are included In the immunogenic compositions. In one preferred embodiment, administration of the immunogenic composition of the invention to a mammal inhibits invasion of the cells of the mammal by Neisseria meningitidis. 
Antibodies which specifically bind to the polypeptides of the invention are provided. In a preferred embodiment, the antibodies bind to a polypeptide such as ORF 1 (SEQ ID NO:2), ORF 2a (SEQ ID NO:4), ORF 2b (SEQ ID NO:5), or ORF 3 (SEQ ID NO:7); without binding to the E coli FtsZ protein, or to the E coli UNK protein. Typically, the antibodies specifically bind to the ORF 1 (SEQ ID NO:2), ORF 2a (SEQ ID NO:4), ORF 2b (SEQ ID NO:5), or ORF 3 (SEQ ID NO:7) proteins.
The invention provides isolated Neisseria meningitidis diplococcus. The diplococcus has a reduced ability to invade tissue culture epithelial cells in vitro as compared to a wild-type Neisseria meningitidis diplococcus and the genome of the isolated Neisseria meningitidis diplococcus has a modification in the region of the genome corresponding to Seq 4 (SEQ ID NO:8). In one embodiment, the isolated Neisseria meningitidis diplococcus comprises a transposon insertion in the region of the genome corresponding to Seq 4 (SEQ ID NO:8).
The invention provides a variety of assays for detecting Neisseria meningitidis, including PCR assays, northern blots, Southern bloc, western blots and ELISA assays. For example, the invention provides PCR reaction mixtures using template nucleic acids which hybridize to Seq 4 (SEQ ID NO:8) under stringent conditions. The mixture has a primer pair which hybridizes to the template nucleic acid, wherein the primers, when hybridized to the template, serve as initiation sites for primer extension by a thermostable polymerase such as taq or vent DNA polymerase. The products of PCR amplification are detected by detecting the amplified nucleic acid products (amplicons) of the PCR reaction.
In several methods relying on nucleic acid hybridization, the detection of a Neisseria meningitidis nucleic acid in a biological sample is performed by contacting a probe nucleic acid to the sample and detecting binding of the nucleic acid to the Neisseria meningitidis nucleic acid. The probe hybridizes to Seq 4 (SEQ ID NO:8), or the complement thereof. Many assay formats are appropriate, including northern and Southern blotting.
In one embodiment, the invention provides methods of inhibiting the invasion of a mammalian cell by Neisseria meningitidis by expressing an anti-sense RNA molecule in the mammalian cell. The antisense RNA molecule hybridizes to a nucleic acid which hybridizes under stringent conditions to a nucleic acid encoded by Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), Seq 3 (SEQ ID NO:7), or Seq 4 (SEQ ID NO:8). Such anti sense molecules optionally comprise catalytic RNA ribonuclease domains, such as those derived from a ribozyme.